Introduction and Objective: Septic shock marks the point in a severe infection when cascading responses overwhelm compensatory mechanisms resulting in overt cardiovascular failure. The appearance of vasopressor requiring hypotension substantially increases the risk of death from infection. Up to 60% of septic shock non-survivors die in refractory shock during the first 7-10 days of illness. In refractory septic shock both vascular relaxation and constriction ultimately become impaired, acute abnormalities analogous to endothelial dysfunction and injury in chronic atherosclerosis. This investigation is exploring the mediators, signal transduction pathways, and underlying mechanisms of endothelial dysfunction and vascular inflammation. Proposed Course of Work: Explore nitric oxide-triggered signal transduction pathways in a human-mouse hybrid endothelial cell line and primary human endothelial cells. Initial studies demonstrate that exogenous nitric oxide inhibits proteosame function and activates p38 MAPK. Other work has demonstrated potentially important interactions between nitric oxide signaling and peroxisome proliferator-activated receptors (PPARs), like nitric oxide, have been associated with endothelial protection and other aspects of vascular health. Nitric oxide was found to activate PPARgamma and thereby regulate downstream target genes that contain PPAR-response elements. Investigate interactions between NOS inhibitors and TNF alpha-stimulated inflammatory responses in human primary pulmonary microvascular endothelial cells. Develop an in vitro model of endothelial cell dysfunction using a RNA-mediated interference (RNAi) approach in primary cells. Endothelial dysfunction has been associated with reduced eNOS expression or function in a wide variety of models and clinical settings including sepsis and atherosclerosis. Gene knockdown will be followed by phenotypic characterization using Western blot, flow cytometry, and oligonucleotide microarrays in the presence and absence of inflammatory-mediator activation (TNFalpha). In parallel experiments, RNAi will be used to knockdown the BMPR2 gene. Loss of BMPR2 function has been linked to primary and secondary pulmonary hypertension, a form of endothelial dysfunction that affects the pulmonary vasculature. At present, it is planned to combine BMPR2 knockdown with eNOS knockdown in a 2 x 2 design followed by phenotypic characterization and expression profiling. Data from this in vitro work will be examined and analyzed in the context of clinical samples from a protocol (with Michael Solomon, M.D.) enrolling patients with primary pulmonary hypertension. Progress: Transfection of monoblastoid U937 cells with human eNOS resulted in a cell line that produced nitric oxide in response to calcium ionophore, but not in the resting state (Blood, 1997). However, after differentiation with phorbol-12-acetate-13-myristate, eNOS expressing cells produced increased amounts of both TNFalpha and reactive oxygen species by mechanisms that were independent of nitric oxide. Neither Nw-methyl-L-arginine, a NOS inhibitor, nor mutation of the L-arginine binding site of eNOS, rendering it incapable of producing nitric oxide, blocked the ability of eNOS to upregulate TNFalpha. Conversely, co-transfection with superoxide dismutare or deletion of the NADPH binding site of eNOS completely prevented eNOS from upregulating TNFalpha production. These results suggested that eNOS can regulate inflammatory responses through both nitric oxide (J Immunol, 1994; J Biol Chem, 1997) and reactive oxygen species-based signal transduction pathways (J Biol Chem, 2000). Superoxide produced by eNOS was shown to upregulate TNFalpha via p42/44 MAPK activation (J Biol Chem, 2001).